1. Field of the Invention
This invention relates to compounds known as ribozymes.
Ribozymes are polynucleotides which "have the intrinsic ability to break and form covalent bonds." Symons, Ann. Rev. Biochem. 61:641 (1992). Of primary interest here are ribozymes which break bonds--that is, which cleave a long polynucleotide strand into two cleavage fragments. The first ribozymes were thought to act only upon RNA, but ribozymes that cleave single-stranded DNA have recently been reported. Cech et al., U.S. Pat. No. 5,180,818, the disclosure of which is incorporated by reference.
Ribozymes are valuable in vivo therapeutic agents that inactivate target RNA or DNA within the cell. In particular, ribozymes are exciting therapeutic candidates for AIDS. In vivo applications of ribozymes have been described in U.S. Pat. No. 5,254,678, U.S. Pat. No. 5,225,337, U.S. Pat. No. 5,168,053, and U.S. Pat. No. 5,144,019, the disclosures of which are incorporated by reference herein.
Ribozymes also can be efficient in vitro experimental reagents akin to restriction endonucleases, giving a researcher the ability to cleave a polynucleotide at a particular site. In vitro applications of ribozymes have been described in, e.g., U.S. Pat. No. 5,225,337, U.S. Pat. No. 5,180,818, U.S. Pat. No. 5,093,246, U.S. Pat. No. 5,037,746, and U.S. Pat. No. 4,987,071, the disclosures of which are incorporated by reference herein.
Ribozymes have the potential to serve as "catalysts" of chemical reactions, either in vitro or in vivo. In general, a catalyst will assist and/or drive the chemical reaction, without itself being altered in the process. After a catalytic event, the catalyst may be regenerated and is able to assist in another round of chemical reaction. Catalytic reactions may be more specifically described by two parameters--the specificity of a catalyst to selectively interact only with a particular substrate molecule, and the relative ability of a catalyst to alter the kinetics or rate at which a chemical reaction proceeds. Thus a ribozyme, like other catalysts such as protein-based enzymes, may be characterized in terms of both its kinetics and its specificity. Particularly useful ribozymes, like protein-based enzymes, will combine the qualities of being able to act rapidly and with good specificity.
2. Description of the Problem
The first ribozyme was described by Thomas Cech and colleagues in 1982, and was isolated from Tetrahymena thermophila. Kruger et al., Cell 31:147 (1982); U.S. Pat. No. 5,180,818; U.S. Pat. No. 5,116,742; U.S. Pat. No. 5,093,246; U.S. Pat. No. 5,037,746; U.S. Pat. No. 4,987,071. The Tetrahymena ribozyme catalyzed the excision of an intervening sequence (termed an IVS or intron) from within its own RNA, and subsequently ligated the two remaining exons. Other ribozymes of this sort, referred to as "Group I introns," were subsequently identified. Symons, Ann. Rev. Biochem., p. 642. A similar class of self-splicing ribozymes have been identified and denominated "Group II introns." Id. Because the cleavage reactions of Group I and Group II ribozymes are intramolecular and result in alteration of the ribozyme itself, they cannot be described as catalytic. These ribozymes may be termed "native" ribozymes.
Another broad class of native ribozymes was discovered amongst various pathogenic plant RNAs. Long and Uhlenbeck, FASEB J. 7:25-30 (1993). Many of these native ribozymes have been described as "hammerhead" ribozymes, in reference to the secondary structure which the ribozymes assume. Symons, Ann. Rev. Biochem., p. 645. Specifically, the hammerhead structure comprises a highly conserved nucleotide sequence in the region of catalytic activity. The catalytic region is substantially single-stranded RNA and is flanked by three regions of helical base-pairing. The endonuclease reaction catalyzed by the hammerhead ribozymes differs from that of the Group I, Group II, and RNAase P ribozymes in that it is a transesterification reaction producing a 5' hydroxyl and a 2',3'-cyclic phosphate. The native hammerhead ribozymes undergo intramolecular cleavage, with only a single turnover for each. Symons, Ann. Rev. Biochem., p. 642.
Native ribozymes having other secondary structures have also been characterized. Hampel et al., Biochemistry 28:4929 (1989), describe a ribozyme which displays a secondary structure referred to as "hairpin." The hairpin structure, like the hammerhead structure, catalyzes cleavage via a transesterification reaction, and with similar stereochemical properties. Symons, Ann. Rev. Biochem., p. 660. Like the hammerhead structure, the hairpin structure contains regions of highly conserved sequences, with the catalytic site in close proximity to a base-paired region. Id. at 661. Other researchers have identified a ribozyme in the Hepatitis Delta Virus (HDV), and have described the structure as an "axehead." Id. at 662-64. It too contains a highly conserved region, and it too contains several base-paired regions in close proximity to a single-stranded catalytic region. Id.
Following the discovery of native, non-catalytic ribozymes, researchers discovered native ribozymes capable of intermolecular cleavage reactions. In 1983, Guerrier-Takada et al. reported that the RNA component of RNAase P could cleave its tRNA substrate, even in the complete absence of protein. Cell 35:849 (1983). Soon thereafter, Cech et al. reported that a fragment of Tetrahymena catalyzed a number of transesterification reactions in a truly catalytic manner. Symons, Ann. Rev. Biochem., p. 642.
Subsequently, Uhlenbeck and colleagues exploited the highly conserved catalytic region and the helical flanking regions of the hammerhead structure to design the first synthetic catalytic ribozyme. Symons, Ann. Rev. Biochem., p. 647. Other examples of synthetic catalytic ribozymes based on the hammerhead structure followed. E.g., U.S. Pat. No. 5,254,678; Jeffries and Symons, Nucl. Acids Res., 17:1371 (1989); and Koizumi et al., FEBS Letters 239:285 (1988). The hairpin structure has been exploited in the formation of a synthetic ribozyme which cleaves HIV-1 RNA. Ojwang et al., Proc. Nat. Acad. Sci. 89:10802 (1992); U.S. Pat. No. 5,144,019. The HDV ribozyme sequence and structure also has been characterized. Perrotta and Been, Biochemistry 31:16-21 (1992); U.S. Pat. No. 5,225,337.
In order to be of practical value, a ribozyme must act intermolecularly on a separate substrate molecule, and remain intact so as to act on subsequent substrate molecules. Ribozymes which perform such intermolecular reactions are termed catalysts, akin to the enzymatic proteins which catalyze myriad chemical reactions within the cell.
Ribozymes, like protein-based enzymes, may be characterized by the kinetic parameters of the reactions that they catalyze. The rate of catalysis may be described by one parameter designated k.sub.cat, otherwise referred to as the "turnover number." That parameter describes the rate of release of the cleaved substrate, and is measured in terms of number of substrate molecules cleaved and released per minute. If this turnover number is low, the reaction as a whole will be slowed. The literature to date for synthetic ribozymes generally reports k.sub.cat values in the range of 0.5-2.1 per minute, Symons, Ann. Rev. Biochem., p. 649, although one group investigating highly modified hammerhead structures, in which the flanking side-arms of the hammerhead are entirely modified to contain DNA rather than RNA, have reported slightly higher turnover rates. Hendry et al., Nucleic Acids Res. 20:5737-41 (1992) (k.sub.cat of 8.9 per minute). These catalytic rates are well below those of many enzymatic proteins, which are more typically in the range of 10-10,000 per minute. Zubay, Biochemistry, at 141. Although one review states that such low turnover rates "rival that of the typical DNA restriction enzymes," Long and Uhlenbeck, FASEB J. at 26, increased turnover rates would be greatly desired by those who would use ribozymes for either in vitro or in vivo uses.
The catalytic rate of ribozymes is further slowed when synthetic ribozymes are designed to incorporate larger regions of ribozyme/substrate base pairing necessary to provide rapid and stable binding in vivo. E.g., Taylor et al., Nucleic Acids Res. 20:4559 (1992); Heidenreich and Eckstein, J. Biol. Chem. 267:1904-1909 (1992); Bennett and Cullimore, Nucleic Acids Res. 20:831-837 (1992); Goodchild and Kohli, Arch. Biochem. Biophys. 284:386-91 (1991). Although such increased base pairing improves the specificity of the ribozyme catalytic reaction, once the substrate is cleaved the larger regions of base pairing inhibit the release of the cleavage fragments. Id. Thus, to date practical in vivo use of ribozymes has been inhibited by a perceived need to trade off specificity and stability, on the one hand, with rapid catalytic reactions, on the other.
Researchers have attempted to increase the in vivo efficacy of ribozymes by chemically modifying their structures to increase resistance to the natural degradative processes within the cell. A review of such modifications is provided by Heidenreich et al., FASEB J. 7:90-96 (1993). Despite some progress in the chemical modification of synthetic ribozymes, their practical usefulness remains limited, in part because of the low turnover number (k.sub.cat) characteristic of the ribozymes known to date. This is particularly true for synthetic ribozymes which have been designed with extensive regions of substrate interaction designed to optimize the specificity of the interaction between synthetic ribozyme and substrate.
Accordingly, there exists a need for synthetic ribozymes having improved stability and rates of catalytic turnover, both for in vitro and in vivo applications.